Age-dependent sex differences in non-stenotic intracranial plaque of embolic stroke of undetermined source

Age and sex have effect on atherosclerosis. This study aimed to investigate their effect on non-stenotic intracranial atherosclerotic plaque (NIAP) in embolic stroke of undetermined source (ESUS) using high-resolution magnetic resonance imaging (HR-MRI). We retrospectively recruited consecutive ESUS patients who underwent intracranial HR-MRI to assess the plaque characteristics (remodeling index [RI], plaque burden [PB], fibrous cap [FC], discontinuity of plaque surface [DPS], intraplaque hemorrhage [IPH] and complicated plaque [CP]). We divided patients into three groups (< 60 years, 60–74 years, ≥ 75 years). 155 patients with ipsilateral NIAP were found from 243 ESUS patients, with 106 men (68.39%) and 49 women (31.61%). In total population or age group under 60 years, there were no significant differences in plaque characteristics between men and women (all p > 0.05). In age group of 60–74 years, men were associated with higher PB (66.27 ± 9.17% vs 60.91 ± 8.86%, p = 0.017) and RI (1.174 vs 1.156, p = 0.019), higher prevalence of DPS (82.50% vs 60.00%, p = 0.036) and complicated plaque (85.00% vs 63.33%, p = 0.036). For subjects ≥ 75 years old, PB were significantly higher in twomen vs men (68.85 ± 6.14% vs 62.62 ± 7.36%, p = 0.040). In addition, the probability for PBupper (≥ median PB), RIupper (≥ median RI) and vulnerable plaque increased as age increased, and its predictive power for index ESUS was higher in men than women. This study identified age-dependent sex differences in NIAP characteristics of ESUS patients, which will help us clarify their etiology.


Study enrollment and information collection
We used the same ESUS cohort, which has been reported in detail in our recent study 15 .We retrospectively recruited patients with acute ischemic stroke in the territory of a unilateral anterior circulation between January 2015 and December 2019.All patients included in the study completed HR-MRI within one week from onset and were eligible for proposed the diagnostic criteria for ESUS.In addition, we further excluded patients with nonstenosing carotid plaque ≥ 3 mm detected by computed tomography angiography (CTA) or carotid ultrasonography, aortic arch atherosclerotic plaque with ulceration or ≥ 4 mm by CTA or transesophageal echocardiogram (TEE), balloon dilatation and stent, previous radiation therapy to head or neck and malignant tumor.The more detailed inclusion/exclusion criteria were reported elsewhere 15 .The study was performed in accordance with the relevant guidelines and regulations, and approved by the Institutional Review Board of General Hospital of Northern Theater Command (IRB: k2019-57), which waived the requirement to obtain informed consent.
According to age, the patients were categorized into three groups: < 60 years group, 60-74 years group, and ≥ 75 years group.The categorization rationale was based on previous studies: (1) a study suggested that plaque prevalence was higher in men than women in most age groups, until the age of 75, when carotid atherosclerosis was more common in women (81.2%) than men (76.5%) 6 ; (2) many studies were grouped by the age of 60 [18][19][20] .

Imaging analysis
In agreement with our previous study 21 , the culprit plaque was defined as a lesion of ipsilateral proximal vascular territory of infarction with clinical symptoms.The detailed image protocol was reported in our recent study 15,21 .As shown in Fig. 1, multidimensional parameters were evaluated using 3.0 T high-resolution magnetic resonance imaging, including plaque remodeling index (RI), plaque burden (PB), fibrous cap (FC), discontinuity of plaque surface (DPS), intraplaque hemorrhage (IPH) and complicated plaque (CP).
The plaque at the maximal luminal narrowing (MLN) site was selected in the cross-sectional images of vessels, while the reference sites were chosen as the neighboring plaque-free or was calculated as the average of the minimal lesion segment proximal and distal to the MLN site due to vessel tapering.The RI was defined as vessel area at the MLN site divided by reference vessel area, and RI upper was defined as the value ≥ median of remodeling index.The PB was calculated as (vessel area luminal area/vessel area at MLN site) × 100%, and PB upper was calculated as the value ≥ median of plaque burden.The thick FC was defined as a continuous band of T2 high signal adjacent to the lumen.DPS was defined as irregularity of plaque luminal surface, such as FC rupture, ulcer plaque, or formation of overlying mural thrombus.IPH was defined as a bright T1 signal ≥ 150% of T1 signal of adjacent muscle or pons.Complicated plaque (CP) was defined as any or both of DPS and IPH based on the definition of a complicated American Heart Association type VI plaque 22 .

Statistical analysis
We used Student's t-test (normally distributed) or the Wilcoxon rank sum test (not normally distributed), as appropriate, for continuous variables, and chi-square test or Fisher's exact for categorical variables, to compare the difference of baseline clinical, plaque characteristics between men and women among different age groups.To explore the age distribution of plaque characteristics across genders, we performed violin plots and percentage bar charts.To assess probability of vulnerable plaque and facilitate statistical analysis, we transformed PB and RI into binary variables by the median, and then we performed probability curves for plaque characteristics.Univariable and multivariable logistic regression analyses were performed to identify whether vulnerable features of plaques were related to mechanism of the ESUS between men and women.All analyses were performed using SPSS version 22, GraphPad software Inc., Prism Version 8 and a p-value (2-tailed) < 0.05 were considered to indicate statistical significance.

Ethics approval
This retrospective study was approved by the Institutional Review Board of General Hospital of Northern Theater Command (IRB: k2019-57).

Results
From the initial cohort of 587 ESUS patients, we excluded patients with bilateral or posterior circulation ESUS (n = 193), carotid plaque of ≥ 3 mm in thickness (n = 91), aortic arch atherosclerosis with ulceration or ≥ 4 mm in thickness (n = 15), paradoxical embolism (n = 27) and poor image quality or incomplete information (n = 18).The final cohort included 243 ESUS patients, of whom 155 (31.6% women) had ipsilateral NIAP and 88 patients had not ipsilateral NIAP.
As shown in Fig. 3, the probability for PB upper and RI upper and vulnerable plaque increased as age increased.Interestingly, the probability of PB upper and RI upper was similar between men and women around age 75.For subjects < 75 years old, the probability of PB upper and RI upperb were higher in men, while the probability of PB upper and RI upperb were higher in women over 75 years old.Furthermore, the probability of DPS, IPH, CP was higher in men regardless of age.In addition, the probability of thick FC was decreased in men as age increased, but increased in women.
Table 2 and Fig. 4 summarizes the sex difference of NIAP ipsilateral vs contralateral to ESUS.The univariable logistic regression analyses adjusting for age or excluding more than 75 years old patients showed that PB, RI, DPS and complicated plaque were related with an index ESUS in total patients or in men (all p < 0.05).In women, only RI was related with an index ESUS (p < 0.05), but the association disappeared after excluding more than 75 years old patients (p = 0.120).Univariable logistic regression analyses were performed to identify the sex difference of ipsilateral vs contralateral NIAP in different age groups (Supplemental Table 4 and Fig. 5).In the age group under 60 years, PB, RI, DPS, and complicated plaque were related with an index ESUS in the overall cohort or in men (p < 0.05), but none of these were related with an index ESUS in women (p > 0.05).In the age group of 60-74 years, RI was related with an index ESUS in total patients (p < 0.05), while PB, RI with an index ESUS in men (p < 0.05), but  www.nature.com/scientificreports/none of these were related with an index ESUS in women.For subjects ≥ 75 years old, RI was related with an index ESUS in the overall cohort, men or women, and PB were related with an index ESUS in total patients or women.On multivariable logistic regression analysis (Table 3), RI was related with an index ESUS in the overall cohort, men or women, and CP was related with an index ESUS in the overall cohort in the age group under 60 years (p < 0.05).In the age group of 60-74 years, RI was related with an index ESUS in the overall cohort  or in men (p < 0.05).For subjects ≥ 75 years old, only RI was related with an index ESUS in the overall cohort (p < 0.05) (Table 3).When combined with PB, RI and complicated plaque for predicting index ESUS (Fig. 6), no significant difference in AUC values was found between men and women (AUC: 0.777 vs o.768) in the age group under 60 years.In the age group of 60-74 years, AUC values were higher in men than women (AUC: 0.710 vs o.617).For subjects ≥ 75 years old, AUC values were higher in women than men, with good predictive power.

Discussion
The current study assesses age-dependent sex differences of intracranial plaque characteristics in ESUS, and identifies significant sex difference of vulnerable plaques characteristics in men vs women, as well as an effect of age on the sex differences in plaque characteristics.We found that there were significant differences in PB, RI, DPS and complicated plaque in women vs men.Men had higher PB and RI, higher prevalence of DPS and complicated plaque.The logistic regression analyses  showed that PB, RI, DPS and complicated plaque were related with an index ESUS in the overall cohort or men, but only RI was related with an index ESUS in women.The results were well in agreement with previous studies in which men tend to have a higher prevalence of plaques with vulnerable features (large intraplaque haemorrhage, thin fibrous cap, large lipid core, more inflammatory cells) than women 6,[23][24][25][26][27] .The gender difference could be related with secretion of estrogen.It is widely accepted that oestradiol has sex-specific protective effects in women against atherosclerosis, a finding which was also confirmed in experimental animal models [28][29][30][31] .
Unfortunately, no estrogen-related information was collected in our study.However thick FC and IPH was not found to be a significant predictor for index ESUS, they were related to atherosclerotic plaque as PB, RI, DPS and complicated plaque.We speculate that the difference may be related to the moderate sample size, and the imbalance of sample size among groups.Furthermore, the sex difference was significantly affected by age in the current study.In the 60-74 year group, the vulnerable characteristics of ipsilateral NIAP were found more frequently in men, however, for patients ≥ 75 years old, the vulnerable characteristics occurred more frequently in women.The results were consistent with previous studies: the Tromsø Study found that carotid plaque prevalence was higher in men than women in most of age groups, but more common in women (81.2%) than men (76.5%) until the age of 75 6 .Also, carotid IPH, an unstable plaque component, was shown to have delayed onset in women 32 .Similarly, coronary histopathology study showed that women over 50 were much more likely to have vulnerable plaque than younger, premenopausal women 33 .The protection of oestradiol diminishes with age after menopause, and there was a marked reduction in circulating oestradiol in postmenopausal women 4 .In addition, no significant differences were found between men and women with regard to plaque characteristics in the age group under 60 years, which may be due to the smaller sample size of women vs men (8 vs 54).
In the present study, we found that the prevalence of DPS, IPH, CP was higher in men regardless of age, which further supported the high prevalence of plaque vulnerability in men.However, the probability of PB upper and RI upper was similar between men and women around age 75.Considered together with the high prevalence of vulnerable characteristics in ≥ 75 years old women, these results could suggest the hypothesis that the high prevalence of plaque vulnerability changed from men to women at this time window.Higher plaque burden, positive remodeling and complicated plaques are more likely linked to an embolic stroke, as the respond to the increased plaque burden, and outward expansion of vessel wall will lead to high risk of vulnerability to rupture causing embolic stroke 34,35 .This could provide a possible explanation for the different proportions of gender at different ages of ESUS.Besides, we also found that plaques characteristics exhibited different predictive powers for ESUS index in men vs women, or among different age groups.Collectively, these results suggest that the differential effect of age and gender on the probability of specific mechanisms underlying ESUS could be considered in future studies.
The strength of this study is that it is the first to assess age-dependent sex difference in intracranial plaque characteristics of ESUS, which could facilitate tailored secondary prevention and clinical trial design for ESUS patients.The main limitations are the retrospective nature, the moderate sample size, and the imbalance of sample size of women vs men, which render this finding from the study less powerful.In addition, the gender difference might be related with secretion of estrogen, but no estrogen-related information was collected in our study.Given the high prevalence of intracranial atherosclerotic disease in Chinese populations, the findings may have limited generalizability to other populations.

Conclusion
This study reported age-related sex differences in the characteristics of intracranial plaques in ESUS patients.The findings could inform secondary prevention strategies and clinical trial design in ESUS.

Data availability
Data are available upon reasonable request.The dataset used and/or analyzed during the current study is available from the corresponding author on reasonable request.

Figure 1 .
Figure 1.Schematic diagram of multidimensional parameters using 3.0 T high-resolution magnetic resonance imaging.Top: A, the plaque with IPH; B, the plaque with a thick FC; C, the plaque with DPS.Bottom: Schematic diagram of PB and remodeling index RI.IPH, intraplaque hemorrhage; FC, fibrous cap; DPS, discontinuity of plaque surface; PB, plaque burden; RI, remodeling index.

Figure 2 .
Figure 2. Age-dependent sex differences in ipsilateral NIAP characteristics.Figure A presented age-dependent sex differences in PB and RI; Figure B presented age-dependent probability of DPS, IPH, thick FC or complicated plaque in male vs female ESUS.NIAP, non-stenotic intracranial atherosclerotic plaque; PB, plaque burden; RI, remodeling index; DPS, discontinuity of plaque surface; FC, thick fibrous cap; IPH, intraplaque hemorrhage; CP, complicated plaque.

Figure 3 .
Figure 3. Probability curves for plaque characteristics in male vs female ESUS.PB upper , the value ≥ median of plaque burden; RI upper , the value ≥ median of remodeling index; DPS, discontinuity of plaque surface; FC, thick fibrous cap; IPH, intraplaque hemorrhage; CP, complicated plaque.

Figure 5 .
Figure 5. Forest plot of association between plaque characteristics and ESUS index among different age groups.PB, plaque burden; RI, remodeling index; DPS, discontinuity of plaque surface; FC, thick fibrous cap; IPH, intraplaque hemorrhage; CP, complicated Plaque.

Figure 6 .
Figure 6.Comparison of ROC analysis of plaque vulnerability among different age groups for predicting ESUS.The model included PB, RI and CP.PB, plaque burden; RI, remodeling index; CP, complicated plaque.